Declercq et al.

Veterinary Research 2013, 44:27

http://www.veterinaryresearch.org/content/44/1/27
VETERINARY RESEARCH

REVIEW Open Access

Columnaris disease in fish: a review with

emphasis on bacterium-host interactions
Annelies Maria Declercq1*, Freddy Haesebrouck2, Wim Van den Broeck1, Peter Bossier3 and Annemie Decostere1

Abstract
Flavobacterium columnare (F. columnare) is the causative agent of columnaris disease. This bacterium affects both
cultured and wild freshwater fish including many susceptible commercially important fish species. F. columnare
infections may result in skin lesions, fin erosion and gill necrosis, with a high degree of mortality, leading to severe
economic losses. Especially in the last decade, various research groups have performed studies aimed at elucidating
the pathogenesis of columnaris disease, leading to significant progress in defining the complex interactions
between the organism and its host. Despite these efforts, the pathogenesis of columnaris disease hitherto largely
remains unclear, compromising the further development of efficient curative and preventive measures to combat
this disease. Besides elaborating on the agent and the disease it causes, this review aims to summarize these
pathogenesis data emphasizing the areas meriting further investigation.

Table of contents 1. The agent

1.1 History and taxonomy
1. The agent Flavobacterium columnare (F. columnare), the causative
1.1 History and taxonomy agent of columnaris disease, belongs to the family
1.2 Morphology and biochemical characteristics Flavobacteriaceae [1-3]. Columnaris disease was first de-
1.3 Epidemiology scribed by Davis among warm water fishes from the
2. The disease Mississippi River [4]. Although unsuccessful in cultivat-
2.1 Clinical signs, histopathology, ultrastructural ing the etiological agent, Davis described the disease and
features and haematology reported large numbers of slender, motile bacteria
2.2 Diagnosis present in the lesions [4]. Upon examining a wet mount
3. Pathogenesis preparation of these lesions, column-like structures
3.1 Colonization formed by these bacteria were evident. The organism
3.2 Exotoxins, bacteriocins and endotoxins was hence named Bacillus columnaris and the disease
3.3 Interaction with the fish immune system elicited columnaris disease. Over the decades the taxo-
4. Importance of environmental factors nomic status of the pathogen has changed several times
5. Experimental trials/pathogenicity tests since the pioneering work of Davis [4]. In 1944, Ordal
6. Control and Rucker were the first to isolate the bacterium from
6.1 Preventive measures a natural outbreak of columnaris disease among sockeye
6.2 Curative approach salmon (Onchorhynchus nerka) [5]. A diluted culture
7. Conclusion medium was used to grow the bacterium. Based on
8. Competing interests cellular morphology, they identified the bacterium as a
9. Authors’ contributions myxobacterium. Organisms classified in the order
10. Acknowledgements Myxobacteria are long, thin Gram-negative rods that
11. References are motile on agar media by a creeping or flexing
motion. They have a life cycle composed of vegetative
* Correspondence: andclerc.declercq@ugent.be
1
cells, microcysts (resting cells), and fruiting bodies,
Department of Morphology, Faculty of Veterinary Medicine,
Ghent University, Merelbeke, Belgium
or only vegetative cells and microcysts [6]. Ordal
Full list of author information is available at the end of the article and Rucker reported that the myxobacterium from
© 2013 Declercq et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
Declercq et al. Veterinary Research 2013, 44:27 Page 2 of 17
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columnaris disease produced both fruiting bodies and
microcysts and named the organism Chondrococcus
columnaris [5]. Garnjobst assigned the bacterium to
the family Cytophagaceae as Cytophaga columnaris
after isolating a pathogenic bacterium resembling
Chondrococcus columnaris morphologically, but not
producing microcysts [7]. Bernardet and Grimont re-
classified the organism and placed it in the family
Cytophagaceae and the genus Flexibacter, assigning it
as Flexibacter columnaris [8]. Finally, in 1996, the bac-
terium received its current name, Flavobacterium
columnare, based on DNA-rRNA hybridization data
and protein and fatty acid profiles [3]. In 1999, the F.
columnare cluster was subdivided in three genomovars
based on differences in 16S rRNA sequences, restriction
fragment length polymorphism (RFLP) and DNA-
DNA-hybridization [9].
1.2 Morphology and biochemical characteristics
The morphological and biochemical characteristics of F.
columnare are summarized in Table 1. For a full bio-
chemical profile of F. columnare, the reader is referred
to Bernardet and Bowman [1].
1.3 Epidemiology
F. columnare is distributed worldwide in fresh water
sources and may infect many different wild and cultured
freshwater fish species, such as (but not limited to) carp,
channel catfish, goldfish, eel, perch, salmonids and til-
apia [1,12-17]. This disease also assails many tropical
freshwater aquarium fish [12,14]. In the channel catfish
(Ictalurus punctatus) industry in the United States, F.
columnare is the second most prevalent bacterium, after
Edwardsiella ictaluri, to cause disease and mortality
[18,19], with yearly losses estimated at 30 million dollars
[20]. This organism can also be part of the bacterial
microbiota of freshwater fish, eggs and the rearing wa-
ters the fish live in [21]. Fish may reside in a clinically
healthy carrier status harbouring an isolate remaining
from a previous outbreak of columnaris disease and in
this way act as an infection source for other fish [22,23].
Fujihara and Nakatani reported that rainbow trout sur-
viving a F. columnare infection can release up to 5 × 103
colony forming units/mL/h of viable bacteria into
tankwater [22]. The gills were shown to be the major
release site of this pathogen. Dead fish would be able to
spread the disease at a higher transmission rate com-
pared to living fish [24].
Several studies have indicated the potential for F.
columnare to survive for extended periods in water. Sur-
vival was demonstrated to be influenced by physical and
chemical characteristics of the surrounding water. Fijan
indicated that F. columnare can survive up to 16 days at
25°C in hard, alkaline water with a high organic load
[25]. Soft water with 10 mg/L CaCO3, especially when
acid or with a low organic content, does not provide a fa-
vorable environment for the organism [25]. Chowdhury
and Wakabayashi determined that calcium, magnesium,
potassium and sodium ions all are important for long-term
survival of F. columnare in water [26]. Ross and Smith
found that survival of F. columnare in static, sterile river
water was directly related to temperature, with a higher sur-
vival percentage at lower temperatures [27]. The bacter-
ium can keep its infectivity in lake water in laboratory
conditions for at least five months [28]. F. columnare is
also capable of surviving in sterile river mud [6]. Ap-
parently, mud slurry often contains sufficient nutrients
to maintain viability of F. columnare longer than sterile
river water. In this case, however, the percentage sur-
vival of F. columnare seeded into mud seems to be
higher at 25°C than at 5°C. Temperatures below 5°C
are even detrimental to F. columnare cells in mud. F.
columnare also grows well on particulate fish feed [29].
When surviving outside the host, F. columnare can
change from a virulent to a less virulent form with an
altered colony morphology, probably to save energy
[28]. It has been suggested that F. columnare strains at
fish farms originate from environmental waters and

Table 1 Morphological and biochemical characteristics of F. columnare (adapted from [1,3,10-12])

Characteristic Description
Growth condition Strictly aerobic
Gram-stain Gram-negative
Morphology Long, slender gliding rods of 4 to 10 μm and 0.3 to 0.5 μm wide. In aging cultures
spheroplasts may occur
Capsule Described to be absent [10] or present [11] depending on the adopted strain
Congo red absorption Present due to an extracellular galactosamine glycan in the mucus and the production of
flexirubin-type pigments
H2S-production Present
Degradation of crystalline cellulose Absent
Degradation of complex acidic polysaccharides of Present
connective tissue
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that the farm environment and practices may select for
virulent strains that cause outbreaks in the farm
[28,30].
2. The disease
2.1 Clinical signs, histopathology, ultrastructural features
and haematology
F. columnare causes acute to chronic infections and typ-
ically affects the gills, the skin and fins. The clinical
manifestation of columnaris disease amongst others is
dependent on the virulence of the eliciting strain. In a
study of Rucker et al., the strains of low virulence in-
duced slow progressive infections at water temperatures
above 21°C and caused massive tissue damage before
death occurred [31]. Strains of high virulence caused ful-
minating infections and killed young salmon (Salmo
salar) in 12 to 24 h at 20°C. Ordinarily, these fish did
not show gross tissue damage at the time of death [31].
The same was found in a study of Pacha and Ordal [32]
and Foscarini [33]. The gross pathology observed in the
fish experimentally infected with strains of F. columnare
of high virulence was usually very limited. Apparently,
death occurred before gross external manifestations of
the disease appeared. However, some of the last fish to
die did show macroscopically visible signs [34]. Besides
the virulence of the strain being a determinant factor, in
coldwater and temperate fish, age also seems to have an
important impact on the severity of the clinical signs. In
young fish, the disease develops acutely and mostly dam-
ages the gills (Figure 1). In adults, the disease may adopt
an acute, subacute or chronic course. When the disease
course is acute or subacute in adult fish, yellowish areas of
necrotic tissue can appear in the gills ultimately resulting in
complete gill destruction (Figure 2) [1,32,35].
In chronic cases, it takes longer before gill damage ap-
pears and skin lesions may develop as well [1,32,35]. On
the body, small lesions start as areas of pale discolor-
ations of the skin, which usually are surrounded by a
zone with a distinct reddish tinge. This mostly begins at
the base of the dorsal fin. Fin deterioration then occurs,
starting from the lesion at the base of the fin and
progressing to the outer edge, the opposite to normal
finrot. The lesions then begin extending laterally from
their common location at the base of the dorsal fin to
Figure 1 F. columnare elicited gill lesions in rainbow trout (Oncorhynchus mykiss) fry (operculum removed). In young fish, the disease is
mostly acute and the gill is the major site of damage. The lesions are exhibited by pale necrotic areas (arrow, bar = 1 cm).
Page 3 of 17
Figure 2 Gill lesions in a shubunkin (Carassius auratus)
(operculum removed) caused by F. columnare. Yellowish-white
areas of degeneration are visible in the ventral part of the first gill
arch (arrow). When the F. columnare infection spreads rapidly
throughout the gill lamellae, the fish may die in a short period of
time without any other apparent lesions. Respiratory distress,
caused by damage to the gills, appears to be the cause of death
(bar = 1 cm).
encircle the fish resembling a “saddle-back” (Figure 3).
The disease therefore is referred to as “saddle-back dis-
ease” [1,32,36]. Finrot is also often present [1,37].
In rainbow trout, the area around the adipose fin may
become dark and show erosions. These lesions expand
to the peduncle, hence the name “peduncle disease” [1].
The lesions may progress cranially and caudally and
even into the deeper skin layers, exposing the muscula-
ture leading to deep ulcers (Figures 4 and 5) [1,35]. The
lesions typically are covered with yellowish-white
mucus [1].
Ulceration of the oral mucosa also occurs, resulting in
mouthrot. These mouth lesions are more lethal than are
the skin lesions, since the painful oral lesions render the
fish anorectic and lead to death due to starvation. More-
over, the disease spreads easily to the mandible and the
maxilla. Secondary infections with fungi or other bac-
teria may deteriorate the situation and can be seen to-
gether with the filamentous bacteria [38]. In tropical
fish, this clinical sign led to the disease being termed
“cotton wool disease” or “mouth fungus” [1]. Finrot can
also be present in tropical fish [37]. Lesions can be
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Figure 3 F. columnare induced a saddleback lesion (arrow) in rainbow trout fry. The lesion is visible as a discoloration starting around its
common location at the base of the dorsal fin and extending laterally to encircle the fish resembling a saddle. Hence, the descriptive term
“saddle-back” is often used and the disease is denoted as “saddle-back disease” (bar = 1 cm).
restricted to local skin discoloration, with or without
ulceration, and degeneration of underlying muscle
fibers [1,12,39].
The skin or gills need to be abraded, for bacteria to
enter the bloodstream and cause systemic infections
[40]. However, Hawke and Thune have isolated F.
columnare from internal organs without any external
lesions appearing [18]. Foscarini described that the
pathological changes to the gill structure caused by
columnaris disease go hand in hand with cardiac alter-
ations [33]. The first day after infection, bradycardia was
noticed with the formation of hyperplastic gill lesions.
Degenerative processes of the lamellae in the following
days resulted in a compensatory tachycardia. This re-
search suggests that the interaction between the im-
paired gill vascular blood circulation and the cardiac
changes could result in the death of the fish [33].
Light microscopic examination of the affected gill
tissue reveals the disappearance of the normal structure of
primary and secondary filaments (Figure 6) [32,33,38,41].
In the initial phase, proliferation of epithelial cells of the gill
filaments can be accompanied by an increase of mucous
cells [33]. The proliferating tissue can occlude the space be-
tween adjacent gill lamellae. In more advanced stages, the
occlusion can be total causing the gill lamellae to be com-
pletely surrounded by the propagating tissue. Congestion of
gill lamellae occurs due to accumulation of blood masses
and inflammatory cell infiltration can be noticed. Edema
causes lifting of the surface epithelium of gill lamellae
from the underlying capillary bed. In more advanced
Figure 4 Skin ulceration (arrow) in a minnow (Phoxinus
phoxinus) caused by F. columnare. The lesion has progressed into
deeper skin layers, exposing the musculature. The edge of the
ulceration displays a distinct reddish tinge and its centre is covered
with yellowish-white mucus (bar = 1 cm).
Page 4 of 17
stages of the disease, fusion of gill lamellae and/or gill
filaments appears [32,33,38,41]. Complete clubbing of
gill filaments can finally result in circulatory failure
and extensive internal hemorrhage [33]. Moreover,
huge clusters of F. columnare can be found on the cell
surface and/or in between necrotic sites (Figure 6,
detail).
Columnaris disease may cause acute ulcerative derma-
titis extending into the hypodermis and the muscle.
Waterlogging can be present. The latter appears when
the osmotic barrier is broken and thus forces water into
the tissues, leading to severe dermal edema. Rupture of
pigment cells with the loss of melanocytes can also be
seen. Columns or hay-stack-like aggregates of bacteria
can be gathered between dermal collagen fibers. The
typical long and slender bacterial cells can easily be
noted upon inspecting hematoxylin and eosin (H&E) or
Giemsa stained sections from affected tissue where they
appear bluish-purple and blue, respectively [38]. When
all these changes occur rapidly, they may proceed to
severe necrosis and sloughing of the epidermis [32,38].
Scanning electron microscopic (SEM) pictures of af-
fected gill arches reveal the presence of rod-shaped
bacterial cells, approximately 0.3-0.5 μm wide and 3-10
μm long (Figure 7). These long, thin bacteria adhere on
the surface of the gills and appear to be aggregated ra-
ther than evenly distributed across the gill epithelium
[42,43]. Transmission electron microscopic (TEM)
examination of gill tissue shows numerous long, slender
bacteria in close contact with the gill tissue (Figure 8).
Figure 5 Tail fin erosion in a minnow caused by F. columnare.
Columnaris disease led to complete disappearance of the upper
half of the fin and exposing the underlying musculature (arrow,
bar = 500 μm).
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Figure 6 Gill section of a koi carp infested with F. columnare. In the left figure, extensive loss of branchial structures is visible. This is an
advanced stage of the disease in which the filaments and the lamellae have fused and the gill epithelium is destroyed (H&E, bar = 100 μm).
Complete clubbing of gill filaments may finally result in circulatory failure and extensive internal hemorrhage. A detail of this is depicted in the
right figure, where F. columnare bacteria are visible as long, slender, purplish structures in between remnants of the gill tissue (H&E, bar = 10 μm).

Bullard and McElwain were the first to describe the ul-
trastructural features of saddleback lesions associated
with experimental infections of F. columnare in channel
catfish and zebrafish (Danio rerio) using SEM [44].
Channel catfish skin lesion samples had margins typified
by shed epidermal cells and lesion centers that exhibited
a multitude of rod-shaped bacterial cells, approximately
3-10 μm long and 0.3-0.5 μm wide, intermixed with cel-
lular debris. Zebrafish skin lesion samples displayed a
multitude of rod-shaped bacterial cells and exhibited
similar ultrastructural changes. Scales were missing or,
when present, denuded of epidermis.
In a cutaneous columnaris challenge model in koi
carp, significant changes in blood parameters were ob-
served in the infected fish [45]. For the hematologic
parameters, a significant decrease was noted in Packed
Cell Volume (PCV), haemoglobin concentration, red
blood cell count, mean corpuscular volume and absolute
lymphocyte counts. As for the biochemical parameters,
marked hyponatremia, hypochloridemia and hypergly-
cemia were observed. Calcium and magnesium levels
dropped only slightly and total serum protein and
albumin-like protein concentrations decreased mildly.
Alkaline phosphatase (ALP), aspartate aminotransferase
(AST), lactate dehydrogenase (LDH) and creatine kinase
(CK) secretions were significantly increased [45]. In a
study by Řehulka and Minařík blood parameters from
brook trout suffering from an acute, natural outbreak of
F. columnare also revealed anemia, but levels of mean
corpuscular volume and mean corpuscular haemoglobin
were higher [15]. Total protein levels fell much below
physiological parameters in infected fish. Calcium con-
centrations were reduced significantly. Blood Urea Ni-
trogen (BUN) measurements were much higher
compared to normal levels. The catalytic activity of AST,
alanine aminotransferases and LDH reached multiples of
normal values. In contrast, ALP concentrations de-
creased. Hypoglycemia was noted. No data were shown
on sodium or chlorine [15].

Figure 8 Gill lamellae of a koi carp following infection by F.

Figure 7 Gill of a rainbow trout infested with F. columnare.
This scanning electron microscopic (SEM) picture of an affected gill
arch reveals the presence of long, thin, rod-shaped bacterial cells,
approximately 0.3-0.5 μm wide and 3-10 μm long (SEM, bar = 5 μm).
columnare. This transmission electron microscopic picture clearly
demonstrates the loss of structure of the gill lamellae. The F.
columnare bacteria are lining up around the remnants of these
lamellae, and are closely associated with the gill epithelium
(TEM, bar = 2 μm).
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2.2 Diagnosis
Timely detection of this pathogenic agent is important
to prevent its spreading and to reduce the economic loss
to fish farmers.
The isolation of F. columnare is possible from external
lesions, provided that the samples are taken from the
edge of recent lesions [1]. F. columnare requires low nu-
trient media. No growth of F. columnare is reported on
trypticase soy agar (TSA), nutrient agar or Marine 2216
agar [1]. In 1944, F. columnare was first cultured on
Cytophaga agar, a nutrient poor medium [5]. Since then,
several media, including Shieh [46] and TYES [47] have
been developed in an attempt to improve growth of the
bacterium. Growth does not occur in media that contain
NaCl concentrations of over 0.5% or that have a pH
lower than six [1]. Depending on the strain, the bacter-
ium grows between 15 and 37°C [9,12], with an optimal
growth occurring between 25°C and 30°C. Colonies
appear after 24 to 48 h of incubation [12]. Glucose does
not improve growth. Considering that F. columnare is
easily overgrown by contaminating bacteria, selective
media have been developed based on inherent resistance
of F. columnare to different antimicrobial agents, in-
cluding polymyxin and neomycin [6,8,25]. Decostere
et al. added one μg/mL tobramycin to modified Shieh
medium, demonstrating this to be an effective selective
supplement for isolating F. columnare [41].
F. columnare largely displays two colony types on solid
media; smooth and rhizoid [48]. Kunttu et al. character-
ized a third, rough colony morphology variant [49]. The
rhizoid colony variants were assigned virulent and mod-
erately adherent, the non-rhizoid rough colony variants
non-virulent and highly adherent, and the smooth col-
ony variants non-virulent and poorly adherent [49].
Colonies of F. columnare are notorious for their some-
times strong adherence to the agar. This trait is also
exhibited in broth cultures where yellow, filamentous
clumps of bacterial cells can form a thick ring at the sur-
face of a glass recipient [12,50]. Adherence may be lost
after several subcultures. Colonies can be recognized by
their distinctive yellow pigmentation. The yellow color is
due to the production of flexirubin pigments [1]. A
method to identify the typical protein profile of F.
columnare is whole-cell protein analysis [3]. Genomovar
ascription has been performed using 16S-restriction
fragment length polymorphism to divide F. columnare
into three genomovars I-III [9]. Compared to RFLP,
single strand conformation polymorphism (SSCP) would
have an improved resolution power in the study of
intraspecies diversity in F. columnare [51]. Polymerase
chain reaction (PCR) has gained interest for definitive
identification of F. columnare based on the selective
amplification of the 16S ribosomal RNA gene using
species-specific primers [52,53]. This technique gives a
Page 6 of 17
conclusive identification of the organism within a few
hours and eliminates the need for biochemical testing
which is laborious and sometimes inconclusive [52].
Besides isolation, other methods may be used for
detecting this pathogen. These include serological methods
such as the enzyme-linked immunosorbent assay [54] and
the fluorescent antibody test [55,56]. Both have proven to
be efficient and rapid for diagnosing columnaris
disease. A loop-mediated isothermal amplification
method (LAMP) for rapid detection of Flavobacterium
columnare from infected fish organs (gills, skin and
head kidneys) was established in channel catfish [57].
PCR may also be adopted, having the advantage of be-
ing able to detect very low levels of F. columnare
[52,53]. In addition, Panangala et al. have developed a
TaqMan-based real-time PCR targeting a 113 bp nu-
cleotide region of the chondroitin AC lyase gene of F.
columnare [58]. This PCR is specific, sensitive and re-
producible for the detection and quantitation of F.
columnare in tissues (blood, gills and kidney) of
infected fish. Moreover, real-time PCR-based methods
are distinctively more advantageous than conventional
PCR as they eliminate the need for detection of ampli-
fied products by gel electrophoresis, thus reducing
costs, time and labour [58]. Length heterogeneity PCR
(LH-PCR) is based on comparing naturally varying
lengths of 16S rDNA PCR products between bacterial
groups [59]. LH-PCR has also proven effective in
detecting F. columnare in fish tissue [60].
3. Pathogenesis
Various reports have noted the difference in virulence
among F. columnare strains [11,12,40,42,61-65]. In the
last decade, various studies have attempted to elucidate
the pathogenesis of columnaris disease. Although sig-
nificant progress has been made, still many questions
remain unanswered on how this pathogenic organism
elicits disease, and information concerning the bac-
teriological events preceding disease and death is
scarce. Li et al. related this lack of knowledge to the ab-
sence of an efficient molecular manipulation system
for F. columnare, especially a plasmid-based inframe
knockout system [66], despite two recent reports on
the establishment of genetic manipulation system for
the bacterium [67,68]. Li et al. identified the type I
restriction-modification system (R-M system) in F.
columnare to improve electroporation efficiency and
suggested that it would be of significant interest to
examine the composition and diversity of R-M systems
in strains of F. columnare in order to set up a suitable
genetic manipulation system for the bacterium [66].
Only very recently, the full genome of F. columnare
ATCC 49512 was sequenced [69].
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3.1 Colonization
The colonization of the fish tissue is to be regarded as a
complex multistep process which can be subdivided into
the stages of attraction, adhesion and aggregation, re-
quiring a step-by-step analysis. The exact factors medi-
ating colonization have however not yet received the
full attention they merit and to date remain largely
unidentified.
The research group of Klesius et al. demonstrated by
means of the traditional capillary tube method that the
mucus from the skin and gills of catfish promotes
chemotaxis of F. columnare [63]. The gliding motility of
this bacterium is well known [1,12,41]. Indeed, the ob-
servation of a drop of bacteria grown in broth under a
phase contrast microscope shows the slowly forward and
backward gliding of F. columnare. Pate and Ordal
described that the location of fibrillar structures span-
ning the gap between the outer membrane and the mu-
copeptide layer might play a role in the gliding motility
of the F. columnare bacterial cells [70]. Although Klesius
et al. acknowledged that they were not able to fully de-
fine the role that chemotaxis plays in the virulence of F.
columnare, the chemotactic response of the more viru-
lent genomovar ll isolates suggested that chemotaxis
could be associated with virulence [63]. LaFrentz and
Klesius developed a culture independent method to
quantify the chemotactic response of F. columnare to
skin mucus using blind-well chemotaxis chambers [71]
generating similar results as stated above [63]. At
least three carbohydrate-binding receptors (D-mannose,
D-glucose and N-acetyl-D-glucosamine) associated with
the capsule of F. columnare might be involved in chemo-
tactic responses [62]. The gliding motility gene gldH was
found to be significantly (P < 0.001) upregulated in F.
columnare as soon as five minutes post-exposure to cat-
fish mucus. When pretreated with D-mannose, there
was no upregulation of gliding motility genes [62].
The ability to adhere is a prerequisite for the success-
ful colonization of the host tissue. Decostere et al.
performed a series of both in vivo and in vitro experi-
ments and found that a highly virulent strain adhered
more readily to the gill tissue of black mollies (Poecilia
reticulata) than did the low virulence strain [11,12,42].
This research group underscored that the adhesion of F.
columnare to the gill tissue constitutes an important
step in the pathogenesis of columnaris disease. Bader
et al. adopted an adhesion defective mutant of F.
columnare in immersion challenge trials and found that
the mortality was reduced with 75% and occurred 24 h
later compared to the strains that still possessed the ad-
hesion capacities, confirming the findings of the former
research group [48]. In channel catfish genomovar II is
considered to be more virulent than genomovar I
[51,72]. Challenge of rainbow trout with genomovar I
Page 7 of 17
and II isolates of F. columnare demonstrated a difference
in the cumulative percent mortalities (CPM), with the
genomovar II isolates inducing significantly higher CPM
[73]. Olivares-Fuster et al. compared adhesion of F.
columnare genomovar I and II strains to the skin and
gill of channel catfish and the gill of zebrafish (Danio
rerio) [43]. At 0.5 h post-challenge, both strains adhered
to the gill of channel catfish at comparable levels, but
significant differences in adhesion were found in later
datapoints over time. They concluded that particular
strains of F. columnare exhibit different levels of adhe-
sion to their fish hosts and that adhesion to fish tissues
is not sufficient to cause columnaris disease. The same
statement was previously made by Klesius et al. [63].
Kunttu et al. have shown that colony morphology
affects the adhesion capacity of F. columnare to polystyr-
ene and put forward the hypothesis that there is a link
between virulence and rhizoid colony morphology
[49,74]. The formation of different colony morphologies
could be caused by changes in the cell surface compo-
nents of the bacteria. It was also found that F. columnare
changes colony morphology during experimental long-
term storage in lake water, indicating the importance of
different colony morphologies to bacterial survival
[64,74,75]. Arias et al. did note that long-term starvation
appears to decrease cell fitness and resulted in loss of
virulence [75]. However, whether adhesion impairment
was the cause of the observed virulence loss was not
investigated.
F. columnare produces two types of mucus. The first
one is an acidic polysaccharide and is made visible by
ruthenium red staining. Another type of mucus is a
basic, partially acetylated polygalactosamine, which can-
not be stained with ruthenium red. Pate and Ordal de-
scribed a capsular material which coated the surface of
the bacterial cell that could be stained with ruthenium
red [70]. They stated that the ruthenium red-positive
material was probably an acid mucopolysaccharide that
might be involved in the adhesive properties of the cells.
The exact role of the mucopolysaccharides was however
not further illuminated in this study. Decostere et al.
demonstrated that the adherence capabilities of a highly
virulent F. columnare strain to the gill were significantly
reduced following treatment of the bacteria with sodium
metaperiodate or incubating them with D-glucose,
N-acetyl-D-glucosamine, D-galactose and D-sucrose
[11]. Treatment with pronase or trypsin did not cause
any significant inhibition of adhesion [11]. The same re-
search group noted that the highly virulent strain had a
thick capsule with a regular and dense appearance, whereas
the capsule of the low virulent strain was much thinner.
This made them to speculate that a lectin-like carbohydrate
substance incorporated in the capsule might be partially
responsible for the adhesion to the gill tissue.
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Sun et al. conducted the first transcriptomic profiling
of host responses to columnaris disease following an
experimental immersion challenge by using illumina-
based RNA-sequencing expression profiling [76]. A
rhamnose-binding lectin (RBL) was detected as by far
the most highly upregulated gene observed in their
differentially expressed set, with expression increasing
105-fold by four hours following infection. This
upregulation dramatically decreased at the later verified
timepoints (24 h and 48 h), suggesting the importance of
this gene during early infection events [76]. Immunohis-
tochemical staining with antisera against an RBL in rain-
bow trout revealed the presence of these RBLs in
mucous cells of the gill and in various cells related to
innate immunity [77]. This expression pattern is consist-
ent with the finding of F. columnare bacterial cell aggre-
gates around goblet cells in common carp [11] and
catfish [43] following experimental challenge with a
highly virulent strain [76]. Beck et al. identified two dis-
tinct catfish families with differential susceptibilities to
columnaris disease, with the one family completely re-
sistant and the other susceptible [78]. In the susceptible
family, an acute and robust upregulation in catfish RBL
was observed following challenge that persisted for at
least 24 h. After exposure of the catfish to different
doses of the putative RBL ligands L-rhamnose and D-
galactose, these sugars were found to protect the fish
against columnaris disease, likely through competition
with F. columnare binding of host RBL. Moreover, RBL
expression was upregulated in fish fasted for 7 d (as
compared to fish fed to satiation daily), yet expression
levels returned to those of satiated fish within 4 h after
re-feeding. These findings highlight putative roles for
RBL in the context of columnaris disease and reveal new
aspects linking RBL regulation to feed availability [79].
Further studies are needed to further pinpoint the (vari-
ous) factor(s) responsible for mediating adhesion to the
fish tissue.
The adhesion to the fish tissue was shown to be im-
pacted by various environmental parameters. Using a gill
perfusion model [61,80], Decostere et al. noted that the
adhesion of a highly virulent strain to the gill tissue was
enhanced by a number of factors, including immersion
of the gill in divalent ion water, the presence of nitrite or
organic matter, and high temperatures [61]. The positive
effect of high temperatures on the adhesion has also
been demonstrated by Kunttu et al. [49]. Adhesion
seems to decrease in vitro as salinity goes up [79]. Ex-
pression of adhesins by bacteria is regulated on two dif-
ferent levels. One is directed by environmental sensing
and transcription of specific regulatory elements, and
the other is a random switching on and off of adhesion
genes by submission of the bacterial population to un-
predictable environmental conditions [81]. If and how
Page 8 of 17
both phenomena are established in F. columnare re-
mains to be elucidated.
An aggregative adhesion pattern of a highly virulent F.
columnare strain onto gill tissue is a distinct feature in both
in vivo and organ culture experiments [35,42,53,61]. This
results in an irregular gill surface covered by a thick mat
consisting of numerous clumps of F. columnare bacterial
cells, most likely impeding oxygen uptake and causing
death of the fish. These microcolonies are not observed
when a low virulence strain is used. Biofilm formation cap-
acity was demonstrated in vitro for one F. columnare strain
when exposed to mucus [67]. These features point towards
biofilm formation potentially being an important stage in
the pathogenicity of F. columnare, warranting its further
investigation.
The developmental switch to the biofilm state is com-
monly regulated by quorum sensing. Quorum sensing
(QS) is a mechanism of gene regulation in which
bacteria coordinate the expression of certain genes in
response to the presence or absence of small signal
molecules. In many Gram-negative bacteria, the signal
molecule is an N-acylhomoserine lactone (AHL) [82].
Additionally, a signaling molecule known as autoinducer-2
(AI-2) may also be employed [83]. Wagner-Döbler et al.
found short-chain AHL-type activity in Flavobacterium sp.,
but no AHL-presence could be confirmed using gas
chromatography-mass spectrometry (GS-MS) [84].
Using light chromatography-mass spectrometry (LC-
MS), Romero et al. described the presence of short-
type AHL activity in the culture media of nine
Tenacibaculum maritimum strains, biofilm-forming
members of the phylum Bacteroidetes, formerly re-
ferred to as “Cytophaga-Flavobacterium-Bacteroides”
group [82]. To our knowledge, so far, QS by AHL or
AI-2 has not yet been demonstrated in F. columnare.
3.2 Exotoxins, bacteriocins and endotoxins
It is known that polysaccharide degradation in combin-
ation with the secretion of various extracellular enzymes
participate in the destruction of skin, muscle and gill
tissue [1], enhancing pathogenicity. In culture, F.
columnare produces an enzyme that degrades chondro-
itin sulfates A and C and hyaluronic acid, the complex
polysaccharides of connective tissue. This so-called
chondroitin AC lyase acts specifically on a group of
acidic mucopolysaccharides found primarily in animal
connective tissue [40]. No correlations were found be-
tween host origin, geographic distribution, and amount
of enzyme produced by different isolates [85]. AC lyase
is alleged to play a role in the virulence of F. columnare
[49,65]. Though high AC lyase activity solely would not
be enough to induce virulence in F. columnare strains,
both high AC lyase activity and gliding motility of the
bacteria would be needed for F. columnare to be virulent
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[49]. Proteases also contribute to damaging the tissue or
enhancing invasive processes [40]. Newton et al. isolated
and partially characterized proteases of 23 isolates of
Flavobacterium columnare derived primarily from chan-
nel catfish raised in the southeastern United States [86].
The bacterial isolates were divided into two groups
according to the apparent molecular masses of proteases
after zymographic resolution by non-reducing, non-
denaturing sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) with gelatin as the prote-
ase substrate. Isolates of group one, produced two pro-
teases with apparent molecular masses of 53.5 and 58
kilodaltons. The isolates of group two, revealed three
proteases with apparent molecular masses of 44.5, 48
and 59.5 kilodaltons. All isolates degraded gelatin and
casein. Seven out of 23 isolates degraded elastin. More
protease was produced in a medium with low nutrients
and salts than in media with higher concentrations of
nutrients. Moreover, a sharp increase was seen in protease
production during the first 24 h of incubation and these
levels dropped only slightly in the remaining days of the
experiment [86].
Bacteria not only need to enter the host tissue, they
also need to eliminate competitive bacteria. Different
strains of F. columnare release specific, non-transmissible,
bactericidal substances equivalent to colicins of Escherichia
coli into the environment to reduce competition from
other bacterial strains [87]. It was postulated that cells of
F. columnare also possess multiple specific receptors for
the bacteriocins, and, consequently, the cells are sensitive
only to those bacteriocins for which the cell possesses
receptors [87]. Antagonism of Pseudomonas sp. MT5
against F. columnare bacteria was found to be very
strong in agar assays [88]. However, antagonistic baths
of the Pseudomonas bacterial strain could not yet prevent
nor treat a F. columnare infection following experimental
challenge [23].
Differences in LPS composition between highly and
low virulent strains of F. columnare retrieved from chan-
nel catfish have been detected [89]. Analysis of LPS by
immunoblotting revealed that an avirulent mutant of a
F. columnare isolate lacked the high molecular weight
components of LPS present in virulent isolates. Based on
these differences of LPS and total protein profiles,
the research group was able to discriminate the attenu-
ated mutant from other F. columnare strains [89].
Kunttu et al. determined LPS-profiles of different colony
morphology variants of F. columnare in rainbow trout
[49]. Colony morphology variants of the same strain
produced a similar single LPS band. However, there were
size differences between different strains. Both research
groups used different LPS extraction and detection
methods, rendering comparison of the obtained results
impossible [49].
Page 9 of 17
3.3 Interaction with the fish immune system
When encountering an infection, the first system activated
is the innate immune system. It is assumed that the surface
mucus layer, as a first physical-immunological barrier, plays
an important adhesive role and that it is part of the innate
host resistance of fish to disease [43]. Antibacterial charac-
teristics of the fish mucus against F. columnare have been
demonstrated. In an experimental challenge trial of cutane-
ous columnaris disease in koi carp [45], lesions were noted
on locations where mucus had been removed. After incu-
bating F. columnare inoculated agar plates, a lower number
of colonies was counted on plates to which mucus was
added. Fluorescent microscopy of a stain-based bacterial
viability assay also revealed a higher number of dead bac-
teria in F. columnare cultured with mucus [45]. Staroscik
and Nelson compared growth, biofilm formation, extracel-
lular protease production and changes in protein expres-
sion of a highly virulent F. columnare strain cultured in
media supplemented with juvenile Atlantic salmon (Salmo
salar L.) skin mucus with the same media without mucus
[67]. Interestingly, and in contrast to the reasoning
resulting from the above mentioned studies, salmon surface
mucus promoted the growth of F. columnare, induced the
bacteria to grow as a biofilm and increased extracellular
protease activity [67]. This might indicate that skin mucus
of different fish species responds differently to the bacteria,
or that it is the F. columnare strain which is critical in
determining the antibacterial capacities of the mucus.
The fish skin in itself also forms a barrier against path-
ogens. Aranishi et al. immersed Japanese eel (Anguilla
japonica) in a F. columnare suspension to monitor der-
mal nonspecific stress responses [90]. They found that
cathepsins B and L activities in the infected fishes
increased more than 1.5-fold over their initial values
over a 48 h period, along with a 4.5-fold increase in
bacteriolytic activity. These cathepsins likely participate
in bacteriolysis associated with Japanese eel skin and
their activities may represent an important nonspecific
response of eels [90].
Some studies reveal that F. columnare might be able
to avoid parts of the immune system. In a study by
Ourth and Bachinski, the catfish alternative complement
pathway (ACP) was inhibited by large amounts of sialic
acid contained by Gram-negative bacterial pathogens,
including F. columnare [10]. Sialic acid seemed to be the
determining factor for the pathogenicity of F. columnare,
as very little or no bactericidal activity was produced
against this bacterium by the catfish ACP. Furthermore,
the latter greatly increased after removal of sialic acid
with neuraminidase [10]. Another recurrent feature in F.
columnare infections, is the lack of an inflammatory re-
sponse as observed upon inspecting affected tissues
microscopically. This resulted in the hypothesis that F.
columnare triggers the endogenous programmed cell
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death machinery of immune cells to evade the immune
system. Do Vale et al. have already proven that apoptosis
can be a very powerful pathogenic strategy by inducing
lysis of phagocytic cells [91]. Sun et al. speculated that
negative regulation of one of the central innate immune
signaling pathways NF-κB, may be the result of immune
evasion or manipulation by F. columnare via secreted
toxins [76]. Furthermore, high levels of inducible nitric
oxide synthases (iNOS), apoptotic-promoting interferon
(IFN) and other members of oxidative stress responses
and apoptotic pathways, such as caspase 8 and G3BP1
(Rasputin), were observed following a F. columnare in-
fection. Rasputin plays an inhibitory role in negative
regulation of apoptosis and was highly downregulated at
all examined time-points [76]. This could explain why
hardly any inflammatory cells appear in early infections
with F. columnare as was described by Morrison et al.
[36].
Ourth and Wilson demonstrated that F. columnare is
resistant to the bactericidal action of serum of non-
immunized catfish via the ACP [92]. Ourth and
Bachinski confirmed this finding [93], but also demon-
strated that the classical, antibody-mediated complement
pathway is highly effective in killing F. columnare [10].
The virulence of F. columnare can also be influenced by
transferrin [94]. The survival time of fish experimentally
challenged with F. columnare by intraperitoneal injec-
tion with iron-free human transferrin (Sigma) was re-
duced when iron was injected prior to exposure. The
effect of iron was only evident in one of the two strains
examined when the challenge was delivered via
immersion. These results indicate that iron depletion
may limit the virulence of F. columnare more in sys-
temic infections than in external infections. This hypoth-
esis is supported by data indicating that administration
of transferrin prior to challenge increased survival after
challenge by injection but had little or no effect on bath-
challenged fish [94]. However, the results found were
not consistent for all strains tested and no statistical data
were presented.
Several immunization experiments adopting different
administration routes, have proven that fish can be
protected from subsequent F. columnare infections by
activating the adaptive immune system [20,22,95-98].
High agglutinin titers and good protection were obtained
in trout following subcutaneous or intraperitoneal injec-
tion with heat-killed F. columnare cells [22]. Schachte
and Mora obtained a high agglutinin titer in channel cat-
fish by intramuscular injection of heat-inactivated cells
of the pathogen, but the actual level of protection was
not examined [99]. Becker and Fujihara reported that
rainbow trout injected with heat-killed cells produced an
agglutinating titer of 1:5120, and that 60-70% of the
trout later survived an injection of 106 live F. columnare
Page 10 of 17
cells [96]. Tilapia (Oreochromis niloticus (L.)) could
mount a significant humoral response in plasma and cu-
taneous mucus to F. columnare after intraperitoneal
immunization with formalin-killed sonicated cells in
Freund’s complete adjuvant [97]. Protection levels were
not investigated in the latter study. Protection was
obtained after oral immunization with heat-killed or
formalin-killed cells of F. columnare in the fish feed of
three-month-old coho salmon (Oncorhynchus kisutch)
[22,100]. However, the protection as observed in these
studies did not go hand-in-hand with high agglutinin ti-
ters. Bath-immunization with a bacterin was shown to
protect carp against experimental challenge but anti-
bodies against F. columnare were not detected in sera
from immunized fish [101]. Song’s work with bacterins
demonstrated unequivocally that immersion vaccination
could result in high levels of protection, but field test re-
sults were inconsistent [102]. Song was able to demon-
strate that there was cross-protection between isolates
and that there may be a common protective antigen
among the strains tested [102]. Polyvalent vaccines have
also been tested. Using intraperitoneal injections of a
combination of formalin-killed F. columnare, Aeromonas
salmonicida and Yersinia ruckeri antigens, interference
from A. salmonicida antigen was shown to suppress re-
sponses to two other antigens [100]. Commercially avail-
able oral and bathing vaccines have been successfully
tested in largemouth bass (Micropterus salmoides) fry
and salmon, respectively [95,103]. Vaccination trials are
further elaborated on below.
4. Importance of environmental factors
Karvonen et al. described the effect of global warming
on the prevalence of different fish parasites and bacteria
[104]. F. columnare could be one of the many taking
advantage of this phenomenon. Indeed, transmission of
columnaris disease is more efficient in higher tempera-
tures [30,105]. Holt et al. found that when steelhead
trout (Salmo gairdneri) or coho salmon (0. kisutch)
experimentally infected with F. columnare were held
in water at 12 to 20°C, mortality increased with
temperature [106]. As stated above, adhesion to gill tis-
sue of a highly virulent F. columnare strain is enhanced
at increased temperature [61] and chondroitin AC lyase
activity of this pathogen increases along with the
temperature [107]. The influence of rearing density and
water temperature in rainbow trout was studied by
Suomalaien et al. [105]. Normal rearing densities with
high temperatures (23°C) proved to increase both trans-
mission rate of columnaris disease and mortality in the
fish. Normal densities at low temperatures (18°C) did
not affect mortality, but increased the transmission rate
of columnaris disease [105].
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Columnaris disease is furthermore influenced by water
quality. Decostere et al. observed significantly higher
bacterial titers on the gills when organic matter or nitrite
were added to an organ bath when performing ex vivo
trials with F. columnare [61]. They discussed that or-
ganic matter could concentrate nutrients to feed the
bacteria and that degrading enzymes could be kept in
close contact with the host tissue. Bacterial titers were
furthermore markedly lower in gills placed in an organ
bath with distilled water with or without 0.03% NaCl
compared to the titers of gills suspended in Ringer solu-
tion or in formulated water containing divalent ions
(magnesium and calcium) [61]. Morris et al. noted that
the survival of the fish exposed to F. columnare signifi-
cantly increased as unionized ammonia concentrations
increased [108]. These results suggest that complex in-
teractions can complicate prediction of the responses of
fish to concurrent chemical stressors and bacterial path-
ogens [108]. Bandilla et al. described that co-infections
of ectoparasites with F. columnare increased the suscep-
tibility of rainbow trout to the bacterial pathogen [109].
Compared with single infections, the mortality was sig-
nificantly higher and the onset of disease condition oc-
curred earlier in fish which were concomitantly infected
by the parasite Argulus coregoni and F. columnare [109].
5. Experimental trials/pathogenicity tests
A reliable and reproducible experimental infection
model is crucial for studying bacterium-host interactions
and evaluating the efficacy of both curative and prevent-
ive measures. Most researchers recognize, in experimen-
tal challenges with F. columnare, the fine and delicate
balance and complex interplay between the bacterial cells,
fish and environment in the successful reproduction of
columnaris disease.
Hitherto, columnaris disease has been reproduced ex-
perimentally in black mollies (Poecilia sphenops) [12],
channel catfish [43,44,110,111], eel [94], golden shiner
(Notemigonus crysoleucas) [112], koi carp [45], rainbow
trout [49,65,105,113], tilapia [94] and zebrafish [43,44].
In the various experimental infection trials, several ways
to both cultivate and harvest the bacterial cells for inocula-
tion were adopted. Different media were used to cultivate
the bacteria, including Hsu-Shotts or modified Hsu-Shotts
medium [79,114], Ordal’s medium [115], Anacker and
Ordal’s medium also referred to as “Cytophaga medium”
[94], F. columnare growth medium broth [115] and Shieh
or modified Shieh medium [12,23,43-45,105]. The bacteria
were mostly grown on a shaker [43-45,75,109-111]. Tem-
peratures and incubation times of the bacteria varied from
21°C to up to 30°C and from 24 to 48 h, respectively.
To reproduce columnaris disease experimentally, largely
two inoculation routes were adopted, viz. bath (immersion)
and injection. With regard to immersion challenges, mostly
Page 11 of 17
full-grown broth cultures were added to the water with
water temperatures of the immersion water varying from
18°C to 30°C [12,23,42-44,111]. Only in a minority of cases
were the bacterial cells first harvested through centrifuga-
tion and consequently resuspended before being added to
the inoculation bath [33,45,114]. Times during which the
fish were bath exposed varied from 15 min [13,72,105] over
30 min [12,23,43-45,109] to 45 min [42], one hour [79,94]
and longer [110,111]. When the bacterial cells were admin-
istered through injection, then they were retrieved from
centrifuged broth cultures following discard of the super-
natant [12] or immediately taken from a full-grown broth
culture without primary centrifugation [13,58,94]. A third
inoculation route consisted of adding dead fish that were
first injected intraperitoneally with a virulent F. columnare
strain, to the aquarium water in which fish were immersed
[20]. In some researches, the successful reproduction of
columnaris disease depended on or the severity of the elic-
ited disease increased by abrading the skin or gills of the
fish [45,94,105] whereas in another study, clipping the fin
had no effect on success after immersion challenge with F.
columnare [12].
The infection route, which determines the way the
bacterial cells are to be grown/collected, also defines the
disease producing capacity of the adopted strains and
the disease picture they elicit. Indeed, especially for low
virulent strains, higher morbidity and mortality rates
were noted after injection of fish as compared to inocu-
lation through immersion [12]. Pacha and Ordal
reported a similar finding [34]. They stated that contact
with highly virulent strains induced infection and disease
contraction more than intramuscular injection, whereas
injection of low virulent bacteria more readily induced
infection and disease contraction than did contact [34].
Environmental conditions can also highly influence
morbidity and mortality rates during a challenge trial.
To illustrate the dramatic effects of water temperature
on the level of mortalities, the investigation of Holt et al.
is particularly relevant [106]. This team challenged steel-
head trout (Oncorhynchus mykiss), Chinook salmon (O.
tshawytscha) and coho salmon with F. columnare, via
the water-borne route. At a water temperature of 9.4°C,
there were no mortalities attributable to F. columnare.
By increasing the temperature to 12.2°C, mortality be-
came as high as 4-20%; at 20.5°C, all the steelhead trout
and coho salmon and 70% of the Chinook salmon died
[106]. High (23°C) rearing temperatures also increased
mortality significantly in rainbow trout compared to
lower (18°C) temperatures [105]. Fish density at fish
farms is also a key player in influencing mortality in an
outbreak of columnaris disease as mortality rates started
earlier and remained higher when fish were stocked at
high densities [105]. Water flow is another important
factor since it acts as a determining factor with regard to
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the contact time between the possibly present bacteria
and the host tissue. High mortality rates were observed in
elvers kept in standing water, while in aquaria with running
water, mortality in elvers was reduced by half [94].
6. Control
6.1 Preventive measures
Management plays a key role in the prevention of the
disease. Cunningham et al. showed that some commonly
recorded production variables (feed consumption, pond
depth, ammonia levels and stocking events) were associ-
ated with columnaris disease outbreaks and, if moni-
tored, could help identify “at risk” ponds prior to disease
outbreaks [116]. Suomalainen et al. pointed out that re-
duction of fish density could be used in the prevention
of columnaris disease especially if water temperature is
high [105]. As lower rearing density can also decrease
the transmission of ectoparasites and penetrating endo-
parasites, it could be an efficient tool in ecological dis-
ease management as a whole [105]. High nitrite levels
and organic load can stimulate the adherence capacity of
F. columnare [61], and therefore it is important to con-
trol these parameters as well. Furthermore, water treat-
ment could aid in averting a bacterial outbreak. Conrad
et al. reported that ozone treatment of water signifi-
cantly reduced the numbers of added F. columnare,
which could be a practical method of prevention [117].
Salt and acidic bath treatments could be used to disin-
fect water contaminated by F. columnare [23]. An
in vivo immersion challenge of F. columnare in chan-
nel catfish and goldfish (Carassius auratus L.) revealed
decreasing mortality as salinity goes up, with signifi-
cantly lower and no mortalities when salinity reaches
values of 1.0‰ and between 3 and 9‰, respectively
[79]. If the fish can be adapted to salt levels of at least
1.0‰, this method could be used as a possible prevent-
ive measure in columnaris disease. Shoemaker et al.
suggested that in the absence of natural food, juvenile
channel catfish should be fed at least once every other
day to apparent satiation in order to maintain normal
physiological function and improve resistance to F.
columnare, since deprivation reduced innate resistance
of catfish to columnaris disease [118].
Besides optimizing and adjusting management prac-
tices, chemical agents can also be adopted as a prevent-
ive approach. Davis concluded that the development or
intensification of columnaris disease could be prevented
by treating the fish for 20 min in a copper sulfate
(CuSO4) bath at 37 mg/L (1:30 000) or by adding copper
sulfate to pond water at 0.5 mg/L [4]. Dipping the fish
one at a time in a 1:2000 copper-sulfate for one to two
minutes was also proven to be effective in the preven-
tion of the disease. Rogers suggested the addition of po-
tassium permanganate (KMnO4) to the water at 2 mg/L
Page 12 of 17
[119]. Darwish et al. also confirmed the prophylactic
value of KMnO4 at doses around 2 mg/L [120].
Prophylactic treatment of channel catfish with 15 mg/L
chloramine-T reduced fish mortality from a F. columnare
infection from 84–100% to 6–14% [121]. Thomas-Jinu
and Goodwin demonstrated the efficacy of prophylactic-
ally given oxytetracycline against mortality in channel cat-
fish and also reported zero mortality for the combination
of sulphadimethoxine and ormetoprim in feed prior to
bacterial challenge with four highly virulent strains of F.
columnare [111].
Another method to prevent columnaris disease is
through vaccination. Although vaccination trials have
not always been successful, success rates have increased
as knowledge on fish immunity and its role in the
defense against bacterial diseases continues to expand.
Bath immunization with a bacterin was shown to protect
carp against experimental challenge, but no agglutinin
could be found in sera from immunized fish [101].
Immersion of channel catfish in a bacterin, when
performed each year, induced a significant decrease in
mortality compared to unvaccinated fish [98]. Fujihara
and Nakatani obtained protection against columnaris dis-
ease in 3-month-old coho salmon by oral immunization
with heat-killed cells of F. columnare incorporated into
fish feed [22]. Ransom proved that prolonged feeding
(over three months) of formalin-killed bacteria provided
high levels of protection [100]. Ourth and Bachinski pro-
posed that strains containing sialic acid could serve as
potential vaccine strains for columnaris disease [10]. As
stated above, immunization with formalin-killed sonicated
cells in Freund’s complete adjuvant injected intraperitone-
ally in tilapia resulted in a significant systemic humoral
response within two weeks and antibody levels almost tri-
pled following secondary immunization [97]. At 10 weeks
postimmunization, the mean antibody titer remained
significantly elevated. Antibodies were also observed in
cutaneous mucus of these fish at six and eight weeks
postimmunization [97]. An attenuated immersion vaccine
currently is registered for the use in channel catfish in the
USA [20]. Fry between 10 to 48 days post hatch that were
vaccinated through immersion achieved a relative percent
survival (RPS) between 57 and 94% following F. columnare
challenge. This vaccine was also proven to be efficient in
largemouth bass fry resulting in RPS values between 74 and
94%, depending on the vaccine dose [20]. Bebak et al. tested
a commercial oral vaccine in largemouth bass fry [95]. Vac-
cinated fish had a 43% lower risk of death by F. columnare
during the field trial [95]. An immersion vaccine consisting
of a bacterin of F. columnare was also brought to the mar-
ket in the USA as an aid in the prevention of columnaris
disease in healthy salmonids of over three grams [103].
Probiotics appear to be a promising way in the preven-
tion of different bacterial diseases in aquaculture [122].
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Boutin et al. isolated different strains of commensal bac-
teria from the skin mucus of unstressed brook charr
(Salvelinus fontinalis) which in vitro revealed antagonis-
tic effects against F. columnare [123]. The strains were
mixed and used to treat columnaris disease in an in vivo
experiment. This resulted in a significant decrease of
mortality indicating the potential use of these probiotic
candidates in the efficient and durable management of
columnaris disease. The immunostimulants β-glucan
and β-hydroxy-β-methylbutyrate raised the levels of im-
mune function parameters, but did not improve survival
in challenge trials with F. columnare at any concentra-
tion of the stimulants used [113]. The research group of
Sink et al. demonstrated that mortality rates in golden
shiners fed high-fat diets with a dairy-yeast prebiotic
were significantly lower after a challenge trial with F.
columnare [112].
Recent studies have demonstrated genetic variation in
resistance towards F. columnare [73,78,124]. Arias et al.
presented experimental data on the susceptibility to
columnaris disease of hybrid catfish (female channel cat-
fish × male blue catfish (I. furcatus)) (C×B) [124]. Under
experimental conditions, C×B hybrids were significantly
more resistant to columnaris disease caused by a highly
virulent strain of F. columnare belonging to genomovar
II compared to channel catfish and blue catfish. Beck
et al. also found one of the two investigated catfish fam-
ilies to be completely resistant towards F. columnare
resulting in no mortality after inoculation with the bac-
teria [78]. These F. columnare resistant families could be
of great financial importance in the catfish industry.
Interestingly, LaFrentz et al. found that the two families
that exhibited the highest CPM after F. columnare
challenges, had the lowest CPM following E. ictaluri
challenge, the latter being the most prevalent bacterium
to cause disease and mortality in the catfish industry
[73]. Further research on larger numbers of families is
needed to determine whether there is any genetic correl-
ation between resistance to E. ictaluri and resistance to
F. columnare, the two leading bacterial diseases in the
catfish industry [125].
6.2 Curative approach
Treatment of columnaris disease using antimicrobial
agents has known different success rates. External treat-
ments are possible only in early stages of the disease,
when the infection is still superficial [6]. Drugs which
have been used effectively in bath therapies are chlor-
amphenicol [126], nifurpirinol [127,128], nifurprazine
[129,130] and oxolinic acid [131,132]. If the disease is
in an advanced stage and/or signs of septicaemia are
observed, it is necessary to administer antimicrobials
in the feed. Oxytetracycline given orally for up to
10 days proved effective in early as well as advanced
Page 13 of 17
outbreaks of columnaris disease in Pacific salmon
(Salmo salar) [6,133]. Lack of success of orally admin-
istered oxytetracycline has also been reported [134].
Sulfonamides, such as sulfamerazine and sulfamethazine,
can be used orally but would be less effective than other
drugs [6,135]. Nitrofuran can also be administered orally
for 3 to 5 days [6,96,130]. Gaunt et al. demonstrated the
efficacy of florfenicol in the feed against columnaris
disease in channel catfish [136]. Darwish et al. also
illustrated the clear benefit of florfenicol against a mixed
infection of A. hydrophila and F. columnare in Sunshine
bass (hybrid striped bass, Morone chrysops female × Morone
saxatilis male) [115]. The excessive use of antimicrobial
agents to withstand F. columnare has its negative attributes
though [20]. These include possible allergic reactions
elicited in the user after food contact [137]. Potential
impacts on human health resulting from the emergence
of drug-resistant bacteria and the associated risk of
transfer of these resistant traits to the environment and
human-associated bacteria are also a major concern [137].
Declercq et al. demonstrated the in vitro multiple resis-
tance of F. columnare strains originating from ornamental
fish toward several clinically important antibiotics, such as
quinolones and tetracyclines [138]. The results obtained
in this study appeal for less prudent use of antimicrobials
especially in the ornamental fish industry and therefore
urges to limit their use and to focus on the development
of alternative curative and preventive measures against
columnaris disease.
Besides resorting to antimicrobial agents, chemicals
have also been adopted in the curative treatment of
columnaris disease. In a study by Thomas-Jinu and
Goodwin, the herbicide DiquatW (Zeneca Agricultural
Products, Wilmington, DE, USA) was shown to signifi-
cantly reduce channel catfish mortalities to zero percent
after challenge with F. columnare [111]. The herbicide
has also proven to be effective in the treatment of
columnaris disease in salmonids [6,139]. Copper sulfate
[4] and potassium permanganate [119] are among the
older chemicals used for treatment and prevention of
columnaris disease in pond fishes. The organic load in
water affects the efficacy of potassium permanganate,
but methods are available to estimate that organic load
and compensate by adjusting the level of the chemical
[6]. Darwish et al. suggested that copper sulfate has clear
therapeutic value against F. columnare infections in
channel catfish when treated in an ultralow flow-
through system during 4 h [110]. Thomas-Jinu and
Goodwin on the other hand proved the inefficacy of this
same chemical against columnaris disease, which might
be due to the advanced stage of the experimental infec-
tion at the time of treatment [111]. An in vitro assay of
commercial products containing peracetic acid was
proven to be effective against F. columnare infection
Declercq et al. Veterinary Research 2013, 44:27
http://www.veterinaryresearch.org/content/44/1/27
[140]. Laanto et al. reported some Flavobacterium sp.
phage lysates to inhibit growth or lyse the bacterial cul-
tures [141]. The authors recommended that the causa-
tive agent of this strong inhibition or lysis should be
studied further for the possibility of developing anti-
microbial agents. Prasad et al. also described the suc-
cessful use of F. columnare phage FCP1 to combat
columnaris disease in walking catfish (Claries batrachus)
[142]. Phage treatment led to disappearance of gross
symptoms, resulted in a negative bacteriological test, a
detectable phage, and 100% survival in experimentally
infected C. batrachus. The result of this study opens new
perspectives for the treatment of columnaris disease elicited
by antimicrobial resistant F. columnare strains [142].
7. Conclusion
Despite the worldwide importance of columnaris disease
and the multitude of studies focusing on its causative
agent, only snippets of the bacterium-host interactions
have hitherto been exposed. This results in major know-
ledge gaps still prevailing on amongst others how the
pathogen is able to establish and maintain a grip on the
skin and gill tissue and elicit disease and mortality.
Methods for rapid identification of the bacterium in the
environment, host tissue or in culture have indeed
evolved rapidly and some techniques permit to trace
even a few bacterial cells. However, the rationale for the
difference in virulence among F. columnare strains re-
mains to be unraveled. Full genome sequencing of the
reference strain has been an important step towards
gaining insight into the pathogenesis of columnaris dis-
ease. Efficient molecular manipulation systems could be
of great importance to further explore this domain.
Genetic knockouts could in effect illuminate virulence
factors involved in the pathogenesis of this disease.
Although various models to experimentally induce
columnaris disease are available, there is a lack of a stan-
dardized experimental model hampering the comparison
of retrieved data in between research groups and the
drawing of consistent conclusions. Consistency in the
cultivation protocol of the bacteria in terms of incuba-
tion temperature, infection dose, exposure time and
infection route is desirable. Extrapolation of a standard-
ized model from one to another fish species is not pos-
sible rendering it necessary to set-up an experimental
model for each fish sensitive species. Besides further elu-
cidating the pathogenesis of columnaris disease, the de-
velopment of efficient curative and effective preventive
measures also needs to continue. With the upcoming
antimicrobial resistance, focus should be laid on the
search for preventive measures. Management plans try-
ing to optimize or adjust fish densities and control water
quality parameters are a first critical step in controlling
columnaris disease. Vaccines, chemotherapeutics and
Page 14 of 17
probiotics could possibly have a promising future in the
prevention and control of the disease as some studies
have already demonstrated. Another interesting envisaged
method to mitigate columnaris disease is the creation of a
fish breeding program introducing F. columnare resistant
fish. Data generated through pathogenesis studies will allow
for the further development and optimization of cost-
effective and environmentally friendly prevention methods
of columnaris disease in the various susceptible fish species.
8. Competing interests
The authors declare that they have no competing interests.
9. Authors’ contributions
AM Declercq: Gathering information and papers, writing the outlines, writing
the paper; A Decostere: Proposing the subject, reviewing and editing the
paper; F Haesebrouck: Critically reviewing the manuscript; W Van den Broeck:
Interpretation of histological, SEM- and TEM- pictures, critically reviewing the
manuscript, P Bossier: Critically reviewing the manuscript. All authors agreed
on outlines and the final version of the paper.
10. Acknowledgements
The authors thank Prof. Paul Simoens of the Department of Morphology of
the Faculty of Veterinary Medicine at Ghent University for critically reviewing
this manuscript. Prof. Koen Chiers of the Department Bacteriology, Pathology
and Avian Diseases of the Faculty of Veterinary Medicine at Ghent University
is gratefully acknowledged for his expertise and help with the interpretation
of the fish pathology. Prof. Maria Cornelissen and Leen Pieters of the
Department of Anatomy, Embryology, Histology and Medical Physics of the
Faculty of Medicine and Health Sciences at Ghent University are accredited
for their cooperation. Furthermore, we also give thanks to Lobke De Bels,
Jurgen De Craene, Bart De Pauw and Patrick Vervaet for their technical
assistance. The financial support by the Special Research Grant (Bijzonder
Onderzoeksfonds, BOF, grant number 01Z06210) of Ghent University,
Belgium is gratefully acknowledged.
Author details
1
Department of Morphology, Faculty of Veterinary Medicine,
Ghent University, Merelbeke, Belgium. 2Department of Pathology,
Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent
University, Merelbeke, Belgium. 3Laboratory of Aquaculture and Artemia
Reference Center, Ghent University, Ghent, Belgium.
Received: 1 February 2013 Accepted: 10 April 2013
Published: 24 April 2013
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doi:10.1186/1297-9716-44-27
Cite this article as: Declercq et al.: Columnaris disease in fish: a review
with emphasis on bacterium-host interactions. Veterinary Research 2013
44:27.